There is a steady increase in the development of microsystems for manipulating, measuring, culturing, and separating cells. Important to the design and adoption of these systems is knowledge of how the systems themselves alter cell state. Such measurements can be difficult to perform, due to either lack of sophistication on the part of the user, or difficulty performing conventional assays with limited numbers of cells in small devices. As a result, assays to measure the impact of microsystems on cell physiology are typically limited to general characteristics such as viability, morphology, and growth. This proposal focuses on developing a suite of cell lines that will provide a more nuanced view of the effects of stress on cell physiology. Building on prior work in our lab creating a heat shock cell sensor, we propose to create a set of cell lines that report on transcriptional activation of stress response pathways relevant to cell-based microfluidic devices. Specifically, we believe that lines reporting on DNA damage, shear stress, and heat shock will provide a valuable set of reagents for designers & users of microsystems. We propose to create these cell lines so that they are spectrally distinct and thus can be mixed and assayed at once. We will formulate a set of standards for applying these reagents and interpreting results from them. As such, we propose to not only generate the cell lines, but the requisite protocols, imaging and data interpretation algorithms, as well as case studies for others to follow. Our specific aims are to (1) create cell lines with inducible fluorescent proteins under the control heat shock-, DNA damage-, and shear stress-inducible promoters. Each cell line will be created in three cell types, representing the diversity of cells one may use in a microsystem; (2) Determine the responses of the cell lines to input stresses, such as shear, heat, and light; (3) Undertake a case study of the use of these reagents and distribute them to the community. PUBLIC HEALTH RELEVANCE: Microsystems that can analyze small numbers of cells could have wide use for point-of- care diagnostics and biotechnology. Currently, there are no standardized metrics by which such microsystems can be designed or used to ensure that they are not harmful to the cells they are trying to analyze. We are proposing to develop cell sensors that would glow colors if they are subjected to stresses from microsystems.